elevated adrenal hsd3b6 underlies salt-sensitive ... · vi, ndei. u, undigested, m, dna marker....

Left Right

CT0

0

3

1

2

4

5

6

7a

b

Supplementary Figure 1 Disorders of Cry-null adrenal glands are bilateral. (a) Bilateral over-production of aldosterone in Cry-null adrenals. The amount of aldosterone, released from tissue culture slices of either left or right adrenal glands were measured as described in Fig. 1c. Plotted are the amounts of aldosterone secreted from WT and Cry-null adrenals at CT0 and CT12. Values are mean ± s.e.m (WT adrenals: CT0, left and right, n=3 each; CT12, left and right, n=6 each; Cry-null adrenals: CT0, left and right, n=3 each; CT12, left and right, n=5 each).*P < 0.05. (b) Bilateral hyperexpression of Hsd3b6 in Cry-null adrenals. Expression of Hsd3b6 in either left or right adrenal glands was analyzed by in situ hybridization. Shown are representative micrographs of WT and Cry-null adrenals, collected at CT0.

Left Right

CT12

Hsd3b6

Left Right Left Right

****

Ald

oste

rone

sec

retio

n (n

g h-

1 pe

r gla

nd)

Cry1–/–Cry2–/–


WT

WT

SUPPLEMENTARY INFORMATION

Elevated adrenal Hsd3b6 underlies salt-sensitive

hypertension in circadian clock-deficient Cry mice

Masao Doi, Yukari Takahashi, Rie Komatsu, Fumiyoshi Yamazaki, Hiroyuki Yamada, Shogo

Haraguchi, Noriaki Emoto, Yasushi Okuno, Gozoh Tsujimoto, Akihiro Kanematsu, Osamu

Ogawa, Takeshi Todo, Kazuyoshi Tsutsui, Gijsbertus T.J. van der Horst & Hitoshi Okamura

Nature Medicine: doi:10.1038/nm.2061

M IU II IIIIVV VI

21 3 4 5 6 7

Whole adrenal cDNA

Supplementary Figure 2 Specification of the Hsd3b subtypes expressed in the Cry-null adrenal gland. Whole adrenal total RNA extract of Cry-null mice was analyzed by RT-PCR using a primer set that can amplify all classes of Hsd3b (sequence similarity enabled the amplification with a single PCR with a common primer set). Aliquots of the PCR products were incubated with isoform specific restriction endonucleases (as determined from the DNA sequence) to diagnose the identity of the PCR products: I, AccI; II, HindIII; III, BstBI; IV and V, AvaI (AvaI digests both IV and V); VI, NdeI. U, undigested, M, DNA marker. Arrowheads indicate the digested Hsd3b6 fragments separated by agarose gel electrophoresis. Note that Hsd3b6 is expressed exclusively in the aldosterone-producing ZG cells (see in situ hybridization of Hsd3b6 in Fig. 2c and Supplementary Fig. 5). Since the ZG cell population only constitutes a fraction of the total number of adrenal gland cells, the concentration of Hsd3b6 transcripts in whole adrenal pool (lane 7) was apparently lower than that of Hsd3b1 (lane 3), which is widely expressed in the adrenal cortex (see Fig. 2c). The types II to V (lanes 4-6) were virtually undetectable.



c

d

BMAL1

CLO

CK

PositiveComplex

NegativeComplex

ROREBmal1

E-boxPer

E-boxCry

E-boxDbp

E-boxRev-erbα

PER

HSD3b6

CRY

CR

Y

DBP

E4BP4 ROREE4bp4

DB

P

DB

P

E4B

P4

E4B

P4

REV-ERB

D-boxHsd3b6

BM

AL1

PE

R

BMAL1

CLO

CK

ROREBmal1

E-boxPer

E-box

E-boxDbp

E-boxRev-erbα

PER

HSD3b6

DBP

E4BP4 ROREE4bp4

DB

P

DB

P

E4B

P4

E4B

P4

REV-ERB

D-boxHsd3b6

BM

AL1

PE

R

WT

Rel

ativ

e m

RN

A le

vels

Rel

ativ

e m

RN

A le

vels

Rel

ativ

e m

RN

A le

vels

Hsd3b6promoter

WT1128bp

WT524bp

M11128bp

M1

M2

M2M1

M21128bp

M1 + M21128bp

0

1

2

3

4

5

Rel

ativ

e lu

cife

rase

act

iviti

es

DBPE4BP4

a

02 6 10 14 18 22

1.0

1.2

1.4

0.2

0.6

0.4

0.8

Rev-erbα

Circadian time

0

0.2

0.6

0.4

0.8

1.0

1.4

1.2

1.6

2 6 10 14 18 22

Bmal1

Circadian time

0

1

0.5

2

1.5

2.5

2 6 10 14 18 22

Clock

Circadian time

0

108642

2018161412

3032

28262422

2 6 10 14 18 22

Per2

Circadian time

0

8

1

4

5

6

7

2

3

2 6 10 14 18 22

Per1

Circadian time

DB

P

DB

P

E4B

P4

E4B

P4

D-box

promoterHsd3b6

+-

+++

++-

--

+-

+++

++-

--

+-

+++

++-

--

+-

+++

++-

--

+-

+++

++-

--

b

Circadian time

02 6 10 14 18 22

1

3

4

2

Dbp

Circadian time

0

1.0

0.5

2.0

1.5

2 6 10 14 18 22

E4bp4

0123456789

1011121314151617

2 6 10 14 18 22Circadian time

WT

WT

Cry1-/-Cry2-/-

Cry1-/-Cry2-/-

Hsd3b6

Supplementary Figure 3 Transcriptional control of Hsd3b6 by the circadian regulators DBP and E4BP4. (a) Promoter analysis of the mouse Hsd3b6 gene. The Hsd3b6 promoter lacks an E-box, but instead contains two functional D-box elements, allowing (i) DBP to activate t r a n s c r i p t i o n a n d ( i i ) E 4 B P 4 t o antagonize DBP-mediated transcription. Transcription assays were performed using H295R cells transiently expressing the luciferase reporter constructs from (mutagenized) promoter regions (as indicated). Error bars indicate the s.e.m from three replicate experiments. (b) Constitutive activation of Dbp in the Cry-null adrenal gland. The circadian expression of Dbp and E4bp4, exhibiting anti-phase rhythms in the WT adrenal gland (black lines), was completely abolished in the Cry-null adrenal gland (red lines). Intriguingly, the expression of Dbp was highly elevated throughout the day in the Cry-null adrenal gland, while E4bp4 was expressed at intermediate levels. Error bars indicate the s.e.m (n= 4, each), normalized to expression of Tbp. (c) Abnormal expression of circadian clock genes in the Cry-null adrenal gland. Similar to Dbp, the expression of the E-box-regulated Per genes was highly elevated in the Cry-null adrenal gland. Relative mRNA levels were determined by qRT-PCR. Error bars indicate the s .e .m (n= 4 , each) , normal ized to expression of Tbp. (d) Schematic model of abnormal control of Hsd3b6 by the circadian regulators DBP and E4BP4. In the WT adrenal gland, transcription of Hsd3b6 is under the well-balanced circadian control by DBP and E4BP4. In the Cry-null adrenal gland, however, constitutive activation of DBP disrupts the equilibrium, leading to imbalanced hyperexpression of Hsd3b6.

-1-1128 -996-1060

-524

Exon1

DBP consensus RTTAYGTAAY WT: -1005 GTTAGGTATT -996 M2: -1005 GGAGACGCTT -996

**** *** *

DBP consensus RTTAYGTAAY WT: -1069 TTTATGTAAA -1060 M1: -1069 TGAGACGCTA -1060

********luciferase

luciferase

luciferase

luciferase

luciferase

The molecular mechanism underlying the abnormal regulation of Hsd3b6 in the ZG cell (Fig. 2) is also worthy of investigation to understand the etiology behind the adrenal-autonomous circadian disorder (Fig. 1). We observed that the gene promoter of Hsd3b6 is controlled by the circadian regulators DBP (ref. 18) and E4BP4 (refs. 20,48). Specifically, the Hsd3b6 promoter contains two functional D-box elements, allowing DBP to activate transcription and E4BP4 to antagonize DBP-mediated transcription (Supplementary Fig. 3a). In the Cry-null adrenal gland the expression of Dbp was constitutively elevated, while E4bp4 remained expressed at intermediate levels (Supplementary Fig. 3b). The constitutively high expression of Dbp represents a feature commonly observed for E-box-controlled genes (Supplementary Fig. 3c,d). Given our previous studies, showing E-box-dependent transcription of Dbp (refs. 18,20), these data favor a scenario in which the DBP-dependent constitutive activation directs the imbalanced super-expression of Hsd3b6 (see model in Supplementary Fig. 3d). It should be noted that although the transcriptional regulation of Hsd3b6 by CRY seems indirect through DBP/E4BP4, it can not be excluded that the activity of the Hsd3b6 promoter might be directly inhibited by CRY in vivo. The latter issue will have to be addressed by a chromatin immunoprecipitation assayusing the adrenal ZG cell.

Cry1–/–Cry2–/–PositiveComplex

NegativeComplex

PositiveComplex

NegativeComplex

PositiveComplex

NegativeComplex


Hsd3b6

24hrExposure time: 2hr

Supplementary Figure 4 Radioisotopic in situ hybridization analysis of Hsd3b6 in the Cry-null adrenal gland. Shown are in situ hybridization micrographs obtained at different exposure times. Shorter exposure results in reduced halation of strong Hsd3b6-positive signals that came from the outer layer of the adrenal cortex.



CZG

ZF

ZR

C

ZG

ZF

Cyp11b2Hsd3b6

Supplementary Figure 5 Expression of Hsd3b6 and Cyp11b2 in the Cry-null adrenal ZG cells. (a) Digoxigenin in situ hybridization analysis of Hsd3b6 and Cyp11b2 in the Cry-null adrenal gland collected at CT0. The layered histoarchitecture of the adrenal: capsule (C); zona glomerulosa (ZG); zona fasciculata (ZF); zona reticularis (ZR). Bar, 50 μm. (b) Double-labeling in situ hybridization of digoxigenin-labeled Hsd3b6 and radioisotope-labeled Cyp11b2 probes. Show is a high magnification view of the region containing ZG cells in the Cry-null adrenal section. Hsd3b6 mRNA-positive cells were colored blue by nitroblue tetraformazan, while Cyp11b2 mRNA signal was shown as isotope-hitted silver grains by emulsion autography. Bar, 25 μm

a b


0

40

20

10

30

50

60

70

85

90

100

Pro

gest

eron

e sy

nthe

sis

(CP

M x

10-

3 )

0

40

20

10

30

50

60

70

85

90

100

CT0 CT12 CT0 CT12

And

rost

ened

ione

syn

thes

is (C

PM

x 1

0-3 )

Supplementary Figure 6 3β-HSD enzymatic activities in the decapsulated adrenal glands of WT and Cry-null mice at CT0 and CT12. The adrenal glands were mechanically separated into the capsular and decapsulated portions. The former portion consists mainly of ZG, while the latter is composed of ZF, ZR, and medulla. Shown are the 3β-HSD activities in the decapsulated portion of the adrenal glands. 3β-HSD activities were evaluated by progesterone synthesis from 3H-pregnenolone (left) and androstenedione synthesis from 3H-dehydroepiandrosterone (right). The synthesized steroid products were counted post fractionation by HPLC. Values are mean ± s.e.m. (n=10–12, each). Note that the 3β-HSD activities in the decapsulated portions of the Cry-null adrenal gland were almost equivalent to those of the WT adrenal gland, while the highly enhanced 3β-HSD activities were observed for the ZG cell-containing capsular portions of the Cry-null adrenal gland (see Fig. 4b).

Cry1–/–Cry2–/–WT


NS HS NS HSDiet:

Supplementary Figure 7 Salt-dependent changes of plasma aldosterone levels in WT and Cry-null mice. Shown are the levels of PAC in WT and Cry-null mice at CT0 after 4-day loading on either a normal salt (NS) or a high salt (HS) diet (n=9–16, each). Values are mean ± s.e.m. *P < 0.05, ***P < 0.001. Note that WT mice experienced a sharp reduction of PAC upon HS loading, while the response of the Cry-null mice was blunted, resulting in retention of excessive PAC even after HS loading.

0

1.5

0.5

1.0

2.0

2.5

3.0

3.5

*

***

Cry1–/–Cry2–/–WT

Pla

sma

aldo

ster

one

(pm

ol m

l-1)


Supplementary Table 1 Subtype specification of Hsd3b

Isoform type

Amplicon size (bp)

Restriction enzyme

Digestion pattern (bp)

I 629 AccI 455 + 174

II 629 HindIII 508 + 121

III 629 BstBI 439 +190

IV and V 629 AvaII 335 + 187 + 107

VI 629 NdeI 462 + 167

Supplementary Table 2 Subtype specification of Cyp11b

Isoform type

Amplicon size (bp)

Restriction enzyme

Digestion pattern (bp)

I 553 NheI 401 + 152

II 556 SacI 390 + 99 +67


Supplementary Table 3

humanhuman

Species

Overview of human and mouse Hsd3b genes

Type Major sites of expressoin Chromosomal localization

II Gonad and adrenal cortex

Gonad and adrenal cortex

Ψ1–5 (pseudogenes)

I

II Kidney and liver

III Kidney and liver

IV Kidney

V Liver

mousemouse

1p12

1

3

p q

HSD3B2

Hsd3b2 Hsd3b1Hsd3b6Hsd3b3

Hsd3b5Hsd3b4

HSD3B1 HSD3BΨ5HSD3BΨ4HSD3BΨ1HSD3BΨ3HSD3BΨ2

1113.3

22.1

22.3

31.2

32.1

32.3

34.1

34.3

36.1

36.3 11 1213.1

21.1

21.212 2221 13.2

22.2

31.1

31.3

32.23334.23536.2

21.3 23 25 32.1

32.3

42.1

42.3 4424 32.231 41 42.2 43

3F2.2

CA1 A3 B D E1 F1 F2.2 F3 H1 H2 H3 H4E3 G1 G3

*

*

I Placenta and skin, plus adrenal ZG

VI Placenta and skin, plus adrenal ZG

Chromosomal localization of the Hsd3b family: The Hsd3b genes in human and mouse form a syntenic cluster at chromosomal position 1p12 and 3F2.2, respectively. The human HSD3B region holds two expressed genes, HSD3B1 (blue arrow) and HSD3B2 (red arrow), and five pseudogenes, Ψ1–5 (black lines). On the other hand, the mouse Hsd3b region contains six expressed genes (types I to VI). The mouse Hsd3b6 gene has been thought to encode a functional homolog of human HSD3B1 (see below). Note that the members of the mammalian Hsd3b family have been chronologically designated according to their order of elucidation in each species. Accordingly, the comparable gene names do not reflect functional similarities (refs 27,28). Information on human and mouse chromosomal DNA sequences were obtained from the Ensembl database (http://www.ensembl.org/index.html).Major expression sites of HSD3Bs in human and mouse: The human HSD3B1 and mouse Hsd3b6 are functionally related because they are both expressed in placenta and skin (refs 27,28). Such expression profiles are unique, as all other Hsd3b isoforms are expressed in non-placental or non-dermal tissues. For example, the human HSD3B2 and mouse Hsd3b1 are expressed in gonad and adrenal cortex, forming another group that is distinct from the human HSD3B1 and mouse Hsd3b6. Moreover, as indicated by the red lines, we observed that human HSD3B1 and mouse Hsd3b6 genes are expressed in the ZG of the adrenal gland (see Figs 2, 3, 6). The subtype-specific tissue distribution profiles seem to be a conserved feature that is common to human and mouse HSD3b genes.


1

Supplementary Methods

Plasma hormone analysis. Plasma aldosterone concentrations were measured using the

Coat-A-Count Aldosteone kit (Siemens International.). This radioimmunoassay (RIA) differs

from other available (commercial) assays in that it does not crossreact with any other steroid,

including corticosterone. The plasma renin activity was determined according to a conventional

RIA procedure (SRL, Tokyo, Japan), which measures the production of angiotensin I from the

endogenous angiotensinogen for a 60-min incubation at 37ºC.

Microarray analysis. Animals were sacrificed by cervical dislocation under a safety red light

at the indicated time points in DD (see Fig. 2a). Total RNA was prepared from two pools of

whole adrenal glands (n = 6, each pool) at each time point to duplicate our observations on two

independent arrays. Total RNA was isolated with the RNeasy mini kit (Qiagen) and the

integrity was assessed by analyzing aliquots on an Agilent 2100 Bioanalyzer (Agilent

Technologies Japan). cDNA synthesis and cRNA labelling were performed using the

One-Cycle Target Labelling and Control Reagents Kit (Affymetrix), and subsequent

hybridization was performed with GeneChip Mouse Genome 430 2.0 array (Affymetrix)

according to the manufacture’ protocol. For data analysis, we used the RMA (robust

multi-array analysis) expression measure48 that represents the log transform of (background

corrected and normalized) intensities of the GeneChips. The RMA measures were computed

using the R package program, which is freely available on the web site

(http://www.bioconductor.org). The value of each time point (Fig. 2a) was expressed as a mean

of the two independent chips, which yielded equivalent results. For each transcript, values were

normalized to the average expression level over the day in the WT adrenal gland. Genes

analyzed in Fig. 2a were following: cytochrome P450, family 11, subfamily a, polypeptide 1

(Cyp11a1); cytochrome P450, family 11, subfamily b, polypeptide 2 (Cyp11b2); cytochrome

P450, family 17, subfamily a, polypeptide 1 (Cyp17a1); cytochrome P450, family 19,

subfamily a, polypeptide 1 (Cyp19a1), cytochrome P450, family 21, subfamily a, polypeptide 1

(Cyp21a1); hydroxysteroid 11-beta dehydrogenase 1 (Hsd11b1); hydroxysteroid (17-beta)

dehydrogenase 1 (Hsd17b1); hydroxysteroid (17-beta) dehydrogenase 2 (Hsd17b2);

hydroxysteroid (17-beta) dehydrogenase 3 (Hsd17b3); hydroxysteroid (17-beta)


2


hydroxysteroid (17-beta) dehydrogenase 7 (Hsd17b7); hydroxysteroid (17-beta)


hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1);







hydroxysteroid dehydrogenase like 1 (Hsdl1); hydroxysteroid dehydrogenase like 2 (Hsdl2);

hydroxysteroid dehydrogenase like 2 (Hsdl2); NAD(P) dependent steroid dehydrogenase-like

(Nsdhl); steroid 5 alpha-reductase 1 (Srd5a1); steroid 5 alpha-reductase 2 (Srd5a2); steroid 5

alpha-reductase 2-like 2 (Srd5a2l2); steroid 5 alpha-reductase 3 (Srd5a3); steroidogenic acute

regulatory protein (Star); StAR-related lipid transfer (START) domain containing 4 (Stard4);

StAR-related lipid transfer (START) domain containing 5 (Stard5); StAR-related lipid transfer

(START) domain containing 6 (Stard6); steroid sulfatase (Sts).

Isoform specification of Hsd3b and Cyp11b. The gene isotypes of Hsd3b and Cyp11b were

identified according to a previously established method39 with slight modifications. In brief, the

amplification of Hsd3b cDNAs was done by PCR with the following primer set: 5’-cag acc atc

cta gat gt-3’ (Forward) and 5’-agg aag ctc aca gtt tcc a-3’ (Reverse), yielding 629-bp DNA

fragments from all known Hsd3b genes (sequence similarity enabled the amplification with a

single PCR with a common primer set). For the amplification of Cyp11b cDNAs, we designed a

primer set: 5’-ctg tca cca aaa gcc gtt c-3’ (Forward) and 5’-ggt tcc gag cca gct ca-3’ (Reverse),

which can amplify the cDNA fragments of both Cyp11b1 and Cyp11b2. The primer sets used

for Hsd3b and Cyp11b were designed to span introns to distinguish mRNA derived cDNA from

amplified genomic DNA. The isotypes of the amplified genes were identified by digestion of

the cDNA sequences with restriction enzymes unique to each of the isotypes. Supplementary

Tables 1 and 2 summarize the restriction enzymes used and the resultant DNA digestion

patterns.


3

Quantitative RT–PCR. Adrenal total RNA was converted to cDNA using SuperScript III

First-Strand Synthesis SuperMix (Invitrogen) and random hexamer primers. Quantitative PCR

analysis of the individual cDNAs was achieved using the Platinum SYBR Green qPCR

SuperMix-UDG (Invitrogen) with StepOnePlus real-time PCR monitoring system (Applied

Biosystems). Results were normalized to Tbp mRNA levels. The primer sets used for the mouse

adrenal study were following: Star (NM_011485), Fw: 5’-ccg gag cag agt ggt gtc a-3’, Rv:

5’-gcc agt gga tga agc acc at-3’; Cyp11a1 (NM_019779), Fw: 5’-aag gta cag gag atg ctg cg-3’,

Rv: 5’-agt gtc tcc ttg atg ctg gc-3’; Hsd3b1 (NM_008293), Fw: 5’-agc atc cag aca ctc tca tc-3’,

Rv: 5’-gga gct ggt atg ata tag ggt a-3’; Hsd3b6 (NM_013821), Fw: 5’-cat cct tcc aca gtt cta

gc-3’, Rv: 5’-tgg tgt gag att aat gta ca-3’; Cyp21a1 (NM_009995), Fw: 5’-gct gtg gct ttc ctg ctt

cac-3’, Rv: 5’-ggc cca gct tga ggt cta act-3’; Cyp11b1 (NM_001033229), Fw: 5’-gtg agc cca tct

tct gac ttt c-3’, Rv: 5’-caa tgt gtc atg agt ggt cat ag-3’; Cyp11b2 (NM_009991), Fw: 5’-gtt ttc

caa tgg tca ctc cag-3’, Rv: 5’-gct tgc tgc ccc tac aaa c-3’; Tbp (NM_013684), Fw: 5’-atg gtg tgc

aca gga gcc aag-3’, Rv: 5’-tca tag cta ctg aac tgc tg-3’; 36B4 (AK010267), Fw: 5’-ctc act gag att

cgg gat atg-3’, Rv: 5’-ctc cca cct tgt ctc cag tc-3’. For the human adrenal study, we assayed

human 36b4 (NM_053275) using the following primer set: 5’-atg cag cag atc cgc atg t-3’ (Fw)

and 5’-ttg cgc atc atg gtg ttc tt-3’ (Rv).

Promoter analysis of Hsd3b6. Luciferase assays with ectopic expression of DBP and E4BP4

were carried out as described20 except that we used aldosterone-producing cell line H295R as a

host. Reporter plasmids used were following: (i) Hsd3b6 promoter-luc: a 1128 bp genomic

DNA fragment corresponding to the 5’-flanking region of mouse Hsd3b6 (-1128 to -1 from the

transcription start site), cloned in pGL3-basic vector (Promega). (ii) M1-luc: Hsd3b6 promoter-

luc mutant in which the sequence of a putative D-box (distal site: -1069 to -1060) was changed

from TGAGACGCTA to TTTATGTAAA. (iii) M2-luc: Hsd3b6 promoter-luc mutant in which

the sequence of a putative D-box (proximal site: -1005 to -996) was changed from

GTTAGGTATT to GGAGACGCTT. (iv) M1+M2-luc: Hsd3b6 promoter-luc mutant in which

the distal and proximal D-box sites were both mutated as indicated in (ii) and (iii). Correct

mutations were all verified by sequencing.

Radioisotopic in situ hybridization. The gene-specific probes used were as follows: for


4

Hsd3b6, the anti-sense probe covering nucleotides 7−310 of the Hsd3b6 mRNA (Genbank,

NM_013821); for Hsd3b1, nucleotides 22−360 (NM_008293); for Cyp11b1, nucleotides

1731−2304 (NM_001033229); for Cyp11b2, nucleotides 1744−2456 (S85260). The

corresponding cDNA fragment was cloned and used as a template for the generation of

riboprobes. The riboprobes were radiolabeled with [33P]UTP (PerkinElmer), using a standard

protocol for the cRNA synthesis. In situ hybridization was performed as described49. Briefly,

paraformaldehyde fixed tissues were frozen and sectioned at a thickness of 30 μm. Then, the

free-floating tissue sections were transferred through 4 x SSC, proteinase K (1 μg ml-1, 0.1 M

Tris buffer [pH 8.0]; 50 mM EDTA) for 15 min at 37°C, 0.25% acetic anhydride in 0.1 M

triethanolamine for 10 min, and 4 x SSC for 10 min. The sections were then incubated in the

hybridization buffer [55% formamide, 10% dextran sulfate, 10 mM Tris-HCl (pH 8.0), 1 mM

EDTA (pH 8.0), 0.6 M NaCl, 0.2% N-laurylsarcosine, 500 μg ml-1 tRNA, 1 x Denhardt’s,

0.25% SDS, and 10 mM dithiothreitol (DTT)] containing radiolabeled riboprobes for 16 h at

60°C. Following a high-stringency posthybridization wash, the sections were treated with

RNase A. Air-dried sections were exposed to X-ray films (Kodak Biomax).

Digoxigenin in situ hybridization. Since digoxigenin-labeled probes allow a better resolution

than isotope probes for analyzing the cellular distribution of mRNA, we made

digoxigenin-labeled antisense cRNA probes using digoxigenin-UTP (Roche Diagnostics)

following a standard protocol of cRNA synthesis. Tissue preparation, prehybridization,

hybridization, and posthybridization washing were the same as for isotope probe hybridization

except that we used 20 μm-thick sections. The sections hybridized with the digoxigenin-labeled

probe were processed for immunochemistry with the nucleic acid detection kit (Roche

Diagnostics). Signals were visualized in a solution containing nitroblue tetrazolium salt (0.34

mg ml-1) and 5-bromo-4-chloro-3-indolyl phosphate toluidinium salt (0.18 mg ml-1) (Roche

Diagnostics).

Double labeling histochemistry for Hsd3b6 and Cyp11b2. Immunocytochemistry of Hsd3b6

was performed using Hsd3b6-specific antibody that we raised in rabbit (see below for details).

Free-floating sections were incubated for 12 h at room temperature with the affinity-purified

Hsd3b6 antibody (3 ng ml-1), followed by incubation for 1 h at room temperature with donkey


5

anti-rabbit IgG, conjugated to red fluorescent dye Alexafluor-594 (1:500 dilution; Invitrogen).

The specificity was confirmed by dilution tests as well as signal absorption using the antigen

peptide. To examine whether the Hsd3b6 is coexpressed with Cyp11b2 in the ZG cells, we

performed double-labeling immunocytochemistry and in situ hybridization. Following the

detection of Hsd3b6 immunofluorescence, sections were processed for the subsequent

digoxigenin in situ hybridization for Cyp11b2. The prehybridization, hybridization, and

posthybridization washes were as described above. The digoxigenin-labeled Cyp11b2 mRNA

signals were stained blue with the nucleic acid detection kit (Roche Diagnostics).

Laser microdissection. Cryosections (5 μm thick) of fresh-frozen adrenal glands were

prepared using a cryostat (Leica) at −17°C and mounted on POL-membrane slides (Leica).

Sections were fixed for 2 min in an ice-cold mixture of ethanol and acetic acid (19:1), rinsed

briefly in ice-cold water, stained for 1 min in ice-cold water containing 0.05% toluidine blue,

followed by two brief washes in ice-cold water (all the solutions were RNase-free). After

wiping off excess water, slides were quickly air dried for 1−2 min at room temperature. As soon

as moistures in the sections decreased enough for laser-cutting, cells in the zona glomurosa and

zona fasciculata were microdissected using a LMD6000 device (Leica; 40× magnification) and

lysed into Trizol reagent (Invitrogen). Upon collection of ∼5000–10,000 cells, total RNA was

purified using the RNeasy micro kit (Qiagen), yielding on average ∼30 ng of RNA. The ratio of

28S:18S rRNA in these RNA samples was approximately 2:1, thus confirming the integrity of

the RNA isolated from laser-microdissected cells. Relative quantification of mRNA was done

by quantitative RT-PCR using the comparative ΔCt method according to a standard protocol

(http://docs.appliedbiosystems.com/pebiodocs/00105622. pdf).

Immunoblotting. Hsd3b6 polyclonal antibody was generated by immunizing rabbit with a

synthetic peptide consisting of amino acid sequence 20−55 of the mouse Hsd3b6 protein, a

region unique to the type VI isoform. The raised antibodies against Hsd3b6 were

affinity-purified using the antigen peptide. A Cyp11a1 (P450scc) antibody was purchased from

Chemicon (rabbit polyclonal, AB1244). Immunoblot analysis was performed using the

following procedure. Adrenal glands, collected at either CT0 or CT12 (n = 4, each), were

homogenized with a glass-Teflon homogenizer in 500 μl of ice-cold 10 mM Tris-HCl (pH 7.6)


6

buffer, containing 0.25 M sucrose and a protease inhibitor cocktail (Roche). The insoluble

debris and nuclei were pelleted by centrifugation at 800g for 10 min. Then, the supernatants

were centrifuged at 105,000g for 60 min (Optima MAX ultracentrifuge, Beckman) to sediment

the fraction containing mitochondria and microsomes. The precipitates were denatured in

Laemmli buffer post measuring the protein concentration by Bradford method. Western

blotting was performed as previously described50. The proteins resolved on SDS-10%

polyacrylamide gel were transferred to a PVDF membrane. The imunoreactivities were

visualized with enhanced chemiluminescence using horseradish peroxidase-conjugatged

anti-rabbit IgG antibody (1:1000, GE healthcare).

Radiotelemetric blood pressure measurement. Arterial blood pressure (BP) was

telemetrically monitored in conscious, freely moving animals with a battery-operated PA-C10

pressure transmitter (Data Sciences International, St Paul, MN). The transmitter was implanted

by surgery under anesthesia with a mixture of ketamine (100 mg kg-1, ip) and xylazine (10 mg

kg-1). The PA-C10 pressure-sensing catheter was inserted into the aortic arch through the left

common carotid artery according to an established protocol51, and the PA-C10 rasiotransmitter

body was placed in a subcutaneous pouch along the animal’s right flank. Following surgery,

animals were returned to their home cages and allowed to recover at least 1 week in LD before

starting data collection in DD. Radio signals from the implanted PA-C10 were captured by

RPC-1 receiver (Data Sciences International, St Paul, MN), and the data were online stored

using the Dataquest ART data acquisition system (Data Sciences International, St Paul, MN).

BP was monitored in 30-sec episodes at 5-min intervals.

48. Irizarry, R.A. et al. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids

Res 31, e15 (2003). 49. Shigeyoshi, Y. et al. Light-induced resetting of a mammalian circadian clock is

associated with rapid induction of the mPer1 transcript. Cell 91, 1043-53 (1997). 50. Doi, M., Okano, T., Yujnovsky, I., Sassone-Corsi, P. & Fukada, Y. Negative control of

circadian clock regulator E4BP4 by casein kinase Iepsilon-mediated phosphorylation. Curr Biol 14, 975-80 (2004).

51. Butz, G.M. & Davisson, R.L. Long-term telemetric measurement of cardiovascular parameters in awake mice: a physiological genomics tool. Physiol Genomics 5, 89-97 (2001).


elevated adrenal hsd3b6 underlies salt-sensitive ... · vi, ndei. u, undigested, m, dna marker....

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